The broad goal of this project is to optimize the ability of zero load rest-intervals to greatly magnify the response of bone to mechanical loading. We identified and developed this novel modification of standard cyclic loading during a previous NIH funded project. Our data indicate that brief rest-intervals between each load cycle serve to lower the magnitude of strain required to initiate periosteal bone formation and diminish the accommodation of bone to repetitive bouts of mechanical loading. In this project, we hypothesize that the osteogenic benefit of rest-inserted loading can be optimized because brief rest-intervals acutely enhance and sustain Ca2+/NFAT mediated gene and protein regulation compared with repetitive cyclic loading. To explore this general hypothesis, we will use an integrated multi-disciplinary approach (in vivo mechanical loading, RT- PCR, immunohistochemistry, histomorphometry, and a novel in silica agent based model of bone cell dynamics) to pursue three S. Aims. In these S. Aims, we will: 1) define an optimal combination of rest-interval duration, days of loading, and cycle number that will maximally enhance periosteal bone formation, 2) define acute alterations in bone cell gene regulation following single bout and multiple bouts of rest-inserted or cyclic loading, and 3) perform a series of in vivo validation experiments that will culminate with an attempt to use an optimized rest-inserted loading intervention to target periosteal bone formation to novel cortical sites in our in vivo model. If we are able to sufficiently understand the underlying signaling pathways of rest-inserted loading such that we are able to optimize and target focal periosteal bone formation, we believe it would then be appropriate to initiate a clinical trial to test this strategy in humans. From a biological perspective, we believe the proposed studies will reveal new insights into the mechanotransduction pathways by which rest-inserted loading derives its substantial benefits. PUBLIC HEALTH RELEVANCE: This project is focused on experimentally optimizing and mechanistically exploring the cellular signaling pathways underlying the surprising effectiveness of zero load rest-intervals in enhancing the response of bone to mechanical loading. From a clinical perspective, success in this project would improve the potential to translate this concept into a non-invasive, non-pharmacologic intervention for bone loss pathologies.